Single-molecule imaging has become a powerful tool for gaining insight into the biochemical activities in living cells, such as DNA bending and tangling within cell nuclei, or transbilayer lipid motion within cell membranes. In some existing methods, optical imaging with resolution beyond the diffraction limit is achieved by localizing single-molecule light emitters, such as fluorescent molecules that may produce a dipole-like radiation pattern and are typically attached to biological structures or other objects of interest.
The fluorescence photons emitted by single molecules contain rich information regarding their rotational motions, but adapting existing methods, such as single-molecule localization microscopy (SMLM), to measure the orientations and rotational mobilities of single-molecule emitters with high precision remains a challenge. Some existing methods have attempted to measure the orientation of these single-molecule emitters to improve the localization accuracy by estimating the localization bias due to orientation effects. Other existing single-molecule imaging methods measure molecular orientation and rotational mobility of single-molecule emitters by modeling the emission patterns of single-molecule emitters or by tuning the excitation polarization. However, these existing methods may require complicated optical instruments and typically lack sufficient sensitivity to measure both 3D molecular orientation and rotational mobility of single molecules simultaneously.